Direct Evidence for Changes in the Resistance of Legume Root Nodules to O2 Diffusion

Indirect evidence suggests that legumes can adjust rapidly theresistance of their root nodules to O₂ diffusion. Here we describeexperiments using O₂ specific micro-electrodes and dark fieldmicroscopy to study directly the operation of this diffusionbarrier. The O₂ concentration sensed by the electrode decreasedsharply in the region of the inner cortex and was less than1.0 mmol m^–3 throughout the infected tissue in nodulesof both pea (Pisum sativum) and french bean (Phaseolus vulgaris).In a number of experiments the ambient O₂ concentration wasincreased to 40% while the electrode tip was just inside theinner cortex. In 13 out of 21 cases the O₂ concentration atthis position either remained low and unchanged or increasedirreversibly to near ambient values. In the remaining casesthe O₂ concentration increased after 1 to 2.5 min and then decreasedto its former value. These results are ascribed to an increasein resistance of the barrier in response to increased O₂ fluxinto the nodule. It was shown microscopically that air spacesboth at the boundary between the infected zone and the innercortex, and within the infected zone started to disappear 3min after nodules were exposed to high ambient O₂ concentrationsand had disappeared completely after 8 min. These spaces werenot changed by exposure of the nodule for 10 min to either N₂or air. Key words: Oxygen, root nodules, air spaces     

Evolution of the 28S ribosomal RNA gene in anurans: regions of variability and their phylogenetic implications

Fifteen restriction sites were mapped to the 28S ribosomal RNA gene of individuals representing 54 species of frogs, two species of salamanders, a caecilian, and a lungfish. Eight of these sites were present in all species examined, and two were found in all but one species. Alignment of these conserved restriction sites revealed, among anuran 28S rRNA genes, five regions of major length variation that correspond to four of 12 previously identified divergent domains of this gene. One of the divergent domains (DD8) consists of two regions of length variation separated by a short segment that is conserved at least throughout tetrapods. Most of the insertions, deletions, and restriction-site variations identified in the 28S gene will require sequence-level analysis for a detailed reconstruction of their history. However, an insertion in DD9 that is coextensive with frogs in the suborder Neobatrachia, a BstEII site that is limited to representatives of two leptodactylid subfamilies, and a deletion in DD10 that is found only in three ranoid genera are probably synapomorphies.      

Human cellular src gene: nucleotide sequence and derived amino acid sequence of the region coding for the carboxy-terminal two-thirds of pp60c-src.

The nucleotide sequence of the 3' two-thirds of a highly conserved, molecularly cloned human cellular src gene (c-src) has been determined. This region of the c-src gene encodes the tyrosine kinase domain of the cellular src protein (pp60c-src) and corresponds to exons 6 through 12 of the chicken c-src gene, as well as nucleotides 545 to 1542 of the Rous sarcoma virus src gene (v-src). The human c-src sequence is very strongly conserved with respect to both the chicken c-src and the Rous sarcoma virus v-src genes, with nearly 90% nucleotide homology observed in this region. Amino acid sequence conservation in this region is even greater; 98% of the amino acids are conserved between human and chicken c-src. Furthermore, the exon sizes and the locations of the exon-intron boundaries are identical in the human and chicken c-src genes. However, sequences within the introns have not been conserved, and the introns within the human c-src gene are significantly larger than the corresponding introns within the chicken c-src gene. The strong amino acid conservation between the carboxy-terminal two-thirds of pp60c-src of species as divergent as humans and chickens suggests that this portion of the pp60c-src protein specifies one or more functional domains that are of great importance to some aspect of normal cellular growth or differentiation.     

Mutants with Altered Ca2+-Channel Properties in PARAMECIUM TETRAURELIA: Isolation, Characterization and Genetic Analysis

Dancers are a group of mutants in Paramecium tetraurelia whose Ca²⁺ current inactivates poorly and are likely to be defective in the structure of their Ca²⁺ channels. These mutants show prolonged backward swimming in response to K⁺ and Ba ²⁺ in the medium and were selected by this property in a galvanotactic trough. The dancer mutants are semidominant, and all isolated mutants belong to one complementation group; they are not allelic to any of the previously isolated behavioral mutants of P. tetraurelia. The phenotypic change from the homozygous parent to heterozygous F₁ generation takes three to five fissions. There is no evidence of a cytoplasmic factor capable of converting the dancer to the wild-type phenotype, as has been demonstrated in the mutants pawn and cnr. We suggest that the dancer locus is a structural gene for the Ca²⁺ channel.     

Direct evidence for an intracellular role for IFN-gamma. Microinjection of human IFN-gamma induces Ia expression on murine macrophages

An intracellular action for IFN-gamma was detected by using microinjection technology. Human IFN-gamma (huIFN-gamma) does not ordinarily act on murine cells because it fails to bind to murine cell surface receptors. However, when huIFN-gamma was microinjected into murine macrophages, a time and dose-dependent induction of Ia was detected by autoradiography on the surface of injected and neighboring cells. These results imply a direct role for internalized IFN-gamma and show that huIFN-gamma, although it fails to be recognized by murine cell surface receptors, can act internally on murine cells. The effect on Ia gene expression induced by microinjected huIFN-gamma was in part indirect: granulocyte/macrophage-CSF (GM-CSF) was released by IFN-gamma-injected macrophages, and this secondary mediator appeared to induce Ia on neighboring cells, inasmuch as anti-GM-CSF blocked Ia induction. Anti-GM-CSF also partially blocked Ia induction by extracellular murine IFN-gamma on murine macrophages. Thus, at least some of the Ia induction attributed to IFN-gamma was mediated by GM-CSF.     

Transformation of chicken embryo fibroblasts by direct DNA transfection of single oncogenes: comparative analyses of src, erbB, myc, and ras.

Chicken embryo fibroblasts (CEF) have been used extensively to study the transformation parameters of a number of avian sarcoma-leukemia viruses. Previously, oncogene transformation of CEF has been conducted almost exclusively with replicating viruses, because of perceived difficulties with direct DNA transfection. Here, we show that CEF can be efficiently and stably transfected by selection for the neomycin resistance gene (neo). Cotransfection of neo with various oncogenes resulted in CEF transformation in vitro and, in several instances, sarcoma formation in vivo. Transfection of src, myc, erbB, and ras, either singly or in combination, resulted in soft-agar colonies with unique morphologies. Transfection of a family of v-src, c-src, and v/c-src chimeric constructs demonstrated the ability of the assay to discriminate between transforming and nontransforming genes. Transfection of a number of erbB variants showed that internal mutations, primarily in the kinase domain, contribute significantly to the ability to transform fibroblasts. The tumorigenic potential detected by transfection of oncogenes faithfully reproduced those previously reported by using viral infections. Our studies establish the utility of CEF transformation by direct DNA transfection. This method should prove useful in analyzing oncogenes, (e.g., myc) that do not readily transform rodent cell lines and in studying host-range mutants of oncogenes, such as those recently identified for src and erbB.